Toxoplasma gondii is an obligate intracellular parasite of humans that can be lethal in immunocompromised patients and the developing fetus. Virulence in T. gondii is driven by secretion of proteins directly into the host cell. The T. gondi secretome is vast and complex, and the majority of the putative secretory proteins are unannotated and their role in T. gondii pathogenesis in unknown. In contrast to T. gondii, Hammondia hammondi and Neospora caninum do not infect humans despite shared gene content, morphology, and life cycle features across these three species. The goal of this project is to identify previously uncharacterized virulence effectors unique to T. gondii. To achieve this goal we will integrate cross-species comparative genomics with functional genomic and forward genetic data to identify candidate effector loci, and then test candidate genes using molecular genetics and infections in vivo. Through whole genome comparisons, we have identified a subset of putative secretory proteins encoded by loci that are uniquely expanded and diversified in T. gondii. These Toxoplasma-Specific Expanded Loci (TSELs) are the focus of this proposal and their importance is confirmed by extensive preliminary data. Specifically, TSEL4 (mitochondrial association factor 1; MAF1) contributes to T. gondii virulence in vivo and is responsible for host mitochondrial association (HMA) with the parasite-containing vacuole. In Aim 1, we will follow up these observations by fully characterizing the MAF1 locus in terms of gene content, diversity, and impact on HMA during infection. We will use targeted deletion and X-ray crystallography to identify the functional domains of MAF1 and to determine its biochemical function. This Aim will be facilitated by our ongoing collaboration with Marty Boulanger (University of Victoria) for structural studies. In Aim 2, we quantify the effect of MAF1 paralog diversification and HMA on host mitochondrial biology. In parallel, we will determine the impact of the same MAF1 paralogs on parasite virulence across the entire T. gondii life cycle (including sporulated oocysts). Collaborations with Bennett Van Houten (University of Pittsburgh Cancer Center) for mitochondrial studies and JP Dubey (USDA) for oocyst production will facilitate this Aim. Finally, in Aim 3 we will characterize 6 additional TSEL identified in our comparative genomic screen. We will determine the extent of locus diversification for each TSEL, and localize individual paralogs in the parasite and host cell. We will also delete each TSEL and determine its impact on T. gondii pathogenesis in vivo. SIGNIFICANCE: Through these studies, we will elucidate how T. gondii manipulation of host mitochondria determines disease severity and outcome. This may reveal novel mitochondrially-driven innate immune pathways since the role of mitochondria in resistance to parasites like T. gondii is largely unknown. This work should also identify T. gondii virulence effectors that may be key virulence determinants in humans. Broadly, this work may also provide new insight into how locus expansion can drive rapid changes in the virulence of any pathogen.